The invention generally relates to vibrational microscopy and imaging systems, and relates in particular to vibrational imaging systems employing Raman scattering.
Conventional vibrational imaging techniques include, for example, infrared microscopy, Raman microscopy, and coherent anti-Stokes Raman scattering (CARS) microscopy.
Infrared microscopy, which generally involves directly measuring the absorption of vibrational excited states in a sample, is limited by poor spatial resolution due to the long wavelength of infrared light, as well as by a low penetration depth due to a strong infrared light absorption by the water in biological samples.
Raman microscopy records the spontaneous inelastic Raman scattering upon a single (ultraviolet, visible or near infrared) continuous wave (CW) laser excitation. Raman microscopy has improved optical resolution and penetration depth as compared to infrared microscopy, but the sensitivity of Raman microscopy is rather poor because of the very low spontaneous Raman scattering efficiency (Raman scattering cross section is typically on the order of 10−30 cm2). This results in long averaging times per image, which limits the biomedical application of Raman microscopy.
CARS microscopy, which uses two pulsed laser beams (pump and Stokes beams), significantly increases the absolute scattering signal due to the coherent excitation. The CARS process, however, also excites a high level of background from the vibrationally non-resonant specimen. Such a non-resonant background not only distorts the CARS spectrum of the resonant signal from dilute sample but also carries the laser noise, significantly limiting the application of CARS microscopy on both spectroscopy and sensitivity perspectives.
One approach to reduce the non-resonant background field in CARS microscopy is to take advantage of the fact that the non-resonant background has different polarization properties than the resonant signal. For example, U.S. Pat. No. 6,798,507 discloses a system in which the pump and Stokes beams are properly polarized and a polarization sensitive detector is employed. Another approach to reducing the non-resonant background field involves detecting the anti-Stokes field in a reverse direction. U.S. Pat. No. 6,809,814 discloses a system in which a CARS signal is received in the reverse direction (epi-direction) from the sample. For transparent samples, however the epi directed signal is significantly smaller than the forward directed signal, and a stronger signal may be desired for certain applications.
There is a need, therefore, for a system and method for providing much improved sensitivity in vibrational imaging, and in particular, to provide a microscopy system that preserves the Raman spectrum (and the associated vibrational signature) and provides an output resonant signal that is readily distinguishable from the non-resonant background.